UBC Theses and Dissertations
Delivery of Messenger RNA to platelets using lipid nanoparticles Novakowski, Stefanie Kim
Platelets are small, anucleate cells that circulate in the blood stream and mediate hemostasis, inflammation, and angiogenesis. Platelet transfusions are used to treat active bleeding as well as to prevent bleeding during thrombocytopenia or prior to surgery. Yet there are situations where transfusions do not adequately stop bleeding, such as during trauma, which is associated with platelet dysfunction. A method for genetically modifying platelets might enhance their efficacy and lead to new therapeutic uses for platelets. As platelets are anucleate, directly modifying platelets requires messenger RNA (mRNA). Attempts to transfect platelets with mRNA have not been successful, and it is unknown whether lipid-based materials could be used as mRNA transfection agents for platelets. Lipid nanoparticles (LNPs) have been used for nucleic acid delivery in vitro and in vivo. In this thesis, the ability of four different classes of LNPs to deliver mRNA to platelets was compared. These classes consisted of LNPs containing cationic lipids (cLNPs) that are highly effective in vitro, LNPs containing ionizable cationic lipids (icLNPs) developed for in vivo use, LNPs without a cationic lipid commonly used to encapsulate proteins or small molecules, and a commercially available agent previously used for short interfering RNA delivery to platelets. To identify ideal conditions for transfection with mRNA, uptake under various storage conditions and the ability of the LNPs to alter platelet activation was quantified. Finally, the ability of platelets to translate and release the mRNA was assessed. Two approaches were taken for mRNA delivery. In one approach, mRNA was synthesized inside of liposomes, indicating proteins, DNA, and small molecules can be delivered to platelets using LNPs. In the second approach, in vitro transcribed mRNA was directly delivered to platelets using icLNPs and cLNPs, and mRNA delivered to platelets using cLNPs was released in microparticles. These were the first examples of direct delivery of mRNA to platelets, and the first step towards creating genetically modified platelets. While protein synthesis in LNP-treated platelets was not detected, optimizing the LNP formulations used here may lead to a transfection agent for platelets that allows for de novo synthesis of exogenous proteins in the future.
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